The present invention provides novel complexes consisting of certain GLP-1 molecules associated with a divalent metal cation that is capable of co-precipitating with a GLP-1 molecule. Pharmaceutical compositions and methods of using such complexes for enhancing the expression of insulin in B-type islet cells is claimed, as is a method for treating maturity onset diabetes mellitus in mammals, particularly humans.

This invention relates to the field of pharmaceutical and organic chemistry and provides novel compounds, and pharmaceutical compositions thereof, which are useful for enhancing the expression of insulin from mammalian pancreatic B-type islet cells and for treating maturity onset diabetes mellitus in a mammal.
The endocrine secretions of the pancreatic islets are under complex control not only by blood-borne metabolites (glucose, amino acids, catecholamines, etc.), but also by local paracrine influences. The major pancreatic islet hormones (glucagon, insulin and somatostatin) interact amongst their specific cell types (A, B, and D cells, respectively) to modulate secretory responses mediated by the aforementioned metabolites. Although insulin secretion is predominantly controlled by blood levels of glucose, somatostatin inhibits glucose-mediated insulin secretory responses. In addition to the proposed interislet paracrine regulation of insulin secretion, there is evidence to support the existence of insulinotropic factors in the intestine. This concept originates from the observations that glucose taken orally is a much more potent stimulant of insulin secretion than is a comparable amount of glucose given intravenously.
The human hormone glucagon is a 29-amino acid peptide hormone produced in the A-cells of the pancreas. The hormone belongs to a multi-gene family of structurally related peptides that include secretin, gastric inhibitory peptide, vasoactive intestinal peptide and glicentin. These peptides variously regulate carbohydrate metabolism, gastrointestinal mobility and secretory processing. The principal recognized actions of pancreatic glucagon, however, are to promote hepatic glycogenolysis and glyconeogenesis, resulting in an elevation of blood sugar levels. In this regard, the actions of glucagon are counter regulatory to those of insulin and may contribute to the hyperglycemia that accompanies Diabetes mellitus [(Lund, P. K., et al., Proc. Natl. Acad. Sci. U.S.A., 79:345-349 (1982)].
Glucagon has been found to be capable of binding to specific receptors which lie on the surface of insulin producing cells. Glucagon, when bound to these receptors, stimulates the rapid synthesis of cAMP by these cells. cAMP, in turn, has been found to stimulate insulin expression [Korman, L. Y., et al., Diabetes, 34:717-722 (1985)]. Insulin acts to inhibit glucagon synthesis [Ganong, W. F., Review of Medical Physiology, Lange Publications, Los Altos, Calif., p. 273 (1979)]. Thus, the expression of glucagon is carefully regulated by insulin, and ultimately by the serum glucose level.
The glucagon gene is initially translated from a 360 base pair precursor to form the polypeptide, preproglucagon [Lund, et al., Proc. Natl. Acad. Sci. U.S.A. 79:345-349 (1982)]. This polypeptide is subsequently processed to form proglucagon. Patzelt, C., et al., Nature, 282:260-266 (1979), demonstrated that proglucagon was subsequently cleaved into glucagon and a second polypeptide. Subsequent work by Lund, P. K., et al., Lopez L. C., et al., Proc. Natl. Acad. Sci. U.S.A., 80:5485-5489 (1983), and Bell, G. I., et al., Nature 302:716-718 (1983), demonstrated that the proglucagon molecule was cleaved immediately after lysine-arginine dipeptide residues. Studies of proglucagon produced by channel catfish (Ictalurus punctata) indicated that glucagon from this animal was also proteolytically cleaved after adjacent lysine-arginine dipeptide residues [Andrews P. C., et al., J. Biol. Chem., 260:3910-3914 (1985), Lopez, L. C., et al., Proc. Natl. Acad. Sci. U.S.A., 80:5485-5489 (1983)]. Bell, G. I., et al., supra, discovered that mammalian proglucagon was cleaved at lysine-arginine or arginine-arginine dipeptides, and demonstrated that the proglucagon molecule contained three discrete and highly homologous peptide molecules which were designated glucagon, glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 2 (GLP-2). Lopez, et al., concluded that glucagon-like peptide 1 was 37 amino acid residues long and that glucagon-like peptide 2 was 34 amino acid residues long. Analogous studies on the structure of rat preproglucagon revealed a similar pattern of proteolytic cleavage between adjacent lysine-arginine or arginine-arginine dipeptide residues, resulting in the formation of glucagon, GLP-1 and GLP-2 [Heinrich, G., et al., Endocrinol., 115:2176-2181 (1984)]. Human, rat, bovine, and hamster sequences of GLP-1 have been found to be identical [Ghiglione, M., et al., Diabetologia, 27:599-600 (1984)].
The conclusion reached by Lopez, et al., regarding the size of GLP-1 was confirmed by the work of Uttenthal, L. O., et al., J. Clin. Endocrinol. Metabol., 61:472-479 (1985). Uttenthal, et al., examined the molecular forms of GLP-1 which were present in the human pancreas. Their research shows that GLP-1 and GLP-2 are present in the pancreas as 37 amino acid and 34 amino acid peptides, respectively.
The similarity between GLP-1 and glucagon suggested to early investigators that GLP-1 might have biological activity. Although some investigators found that GLP-1 could induce rat brain cells to synthesize cAMP [Hoosein, N. M., et al., Febs Lett. 178:83-86 (1984)], other investigators failed to identify any physiological role for GLP-1 (Lopez, L. C., et al.). The failure to identify any physiological role for GLP-1 caused some investigators to question whether GLP-1 was in fact a hormone and whether the relatedness between glucagon and GLP-1 might be artifactual.
Variants of GLP-1 (7-37) and analogs thereof, also have been disclosed. These variants and analogs include, for example, Gln9-GLP-1 (7-37), D-Gln9-GLP-1 (7-37), acetyl-Lys9-GLP-1 (7-37), Thr16-Lys18-GLP-1(7-37), Lys18-GLP-1 (7-37) and the like, and derivatives thereof including, for example, acid addition salts, carboxylate salts, lower alkyl esters, and amides [see, e.g., WO 91/11457]. Generally, the various disclosed forms of GLP-1 are known to stimulate insulin secretion (insulinotropic action) and cAMP formation [see, e g., Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)].
More importantly, multiple authors have demonstrated the nexus between laboratory experimentation and mammalian, particularly human, insulinotropic responses to exogenous administration of GLP-1, particularly GLP-1 (7-36)NH2 and GLP-1 (7-37) [see, e.g., Nauck, M. A., et al., Diabetologia, 36:741-744 (1993); Gutniak, M., et al., New England J. of Medicine, 326(20):1316-1322 (1992); Nauck, M. A., et al., J. Clin. Invest., 91:301-307 (1993); and Thorens, B., et al., Diabetes, 42:1219-1225 (1993)].
More particularly, the fundamental defects identified as causing hyperglycemia in maturity onset diabetes are impaired secretion of endogenous insulin and resistance to the effects of insulin by muscle and liver [Galloway, J. S., Diabetes Care, 13:1209-1239, (1990)]. The latter defect results in excessive production of glucose from the liver. Thus, whereas a normal individual releases glucose at the rate of approximately 2 mg/kg/minute, in patients with maturity onset diabetes, this amount usually exceeds 2.5 mg/kg/minute resulting in a net excess of at least 70 grams of glucose per 24 hours. The fact that there exists exceedingly high correlations between hepatic glucose production, the fasting blood glucose and overall metabolic control as indicated by glycohemoglobin measurements [Galloway, J. A., supra; and Galloway, J. A., et al., Clin. Therap., 12:460-472 (1990)], it is readily apparent that control of the fasting blood glucose is a sine quo non for achieving overall normalization of metabolism sufficient to prevent the complication of hyperglycemia. In view of the fact that present forms of insulin rarely normalize hepatic glucose production without producing significant hyperinsulinemia and hypoglycemia (Galloway, J. A., and Galloway, J. A., et al., supra) alternative approaches are needed.
Intravenous infusions of GLIP-1 (7-36)NH2 to produce twice normal serum concentrations have been demonstrated to produce the effects indicated in the table below:
However, the long-term stability of GLP-1, particularly GLP-1 as a component of a pharmaceutical composition for administration to mammals, is questionable. In fact, when stored at the low temperature of 4xc2x0 C., by-products of GLP-1 (7-37) have been found as early as eleven months after sample preparation (Mojsov, S., supra). Thus, there exists a need for a more stable GLP-1 compound which can safely be administered to mammals in need of such treatment.
Furthermore, the biological half-life of GLP-1 molecules, particularly those molecules which are affected by the activity of dipeptidyl-peptidase IV (DPP IV) is quite short. For example, the biological half-life of GLP-1 (7-37) is a mere 3 to 5 minutes (U.S. Pat. No. 5,118,666), and is further influenced by its rapid absorption following parenteral administration to a mammal. Thus, there also exists a need for a GLP-1 compound which delays absorption following such administration.
Accordingly, the present invention provides compounds which satisfy the aforementioned stability requirements. The compounds of the present invention also provide delayed absorption following parenteral administration and, consequently, should have extended biological half-lives. Also provided are pharmaceutical compositions of the compounds of the present invention, as well as methods for using such compounds.
The present invention provides a complex consisting of a divalent metal cation associated with and capable of co-precipitating with a compound of the formula:
R1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R2
wherein:
R1is selected from the group consisting of L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, xcex2-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine;
X is selected from the group consisting of Ala, Gly, Val, Thr, Ile, and alpha-methyl-Ala;
Y is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly;
Z is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly;
R2 is selected from the group consisting of NH2, and Gly-OH; providing that the compound has an isoelectric point in the range from about 6.0 to about 9.0 and further providing that when R1 is His, X is Ala, Y is Glu, and Z is Glu, R2 must be NH2.
Also provided by the present invention is a pharmaceutical composition comprising a compound of the present invention in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients.
The present invention further provides a method for enhancing the expression of insulin comprising providing to a mammalian pancreatic B-type islet cell an effective amount of a compound of the present invention, as well as a method of treating maturity onset diabetes mellitus which comprises administering to a mammal in need of such treatment an effective amount of a compound of the present invention.
One aspect of the present invention provides a complex consisting of a GLP-1 molecule having an isoelectric point in the range from about 6.0 to about 9.0, complexed with a divalent metal cation.
As used in the present specification, the term xe2x80x9cGLP-1 moleculexe2x80x9d refers to naturally-occurring GLP-1 (7-36)NH2, GLP-1 (7-37), natural and unnatural functional analogs and derivatives thereof, and salts thereof. The amino acid sequence of GLP-1 (7-36)NH2 is well known in the art, but is presented below as a convenience to the reader (SEQ ID NO: 1)
His7-Ala-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20-Glu-Gly-Gln-Ala-Ala25-Lys-Glu-Phe-Ile-Ala30-Trp-Leu-Val-Lys-Gly35-Arg-NH2.
For GLP-1 (7-37), the carboxy-terminal amide functionality of Arg36 is displaced with Gly at the 37th position of the GLP-1 (7-36)NH2 molecule.
In addition, the existence and preparation of a multitude of protected, unprotected, and partially protected natural and unnatural functional analogs and derivatives of GLP-1 (7-36)NH2 and GLP-1 (7-37) molecules have been described in the art [see, e.g., U.S. Pat. Nos. 5,120,712 and 5,118,666, which are herein incorporated by reference, and Orskov, C., et al., J. Biol. Chem., 264(22):12826-12829 (1989) and WO 91/11457 (Buckley, D. I., et al., published Aug. 8, 1991)].
As known in the art, amino acid residues may be in their protected form in which both amino and carboxy groups possess appropriate protecting groups, partially-protected form in which either amino or carboxy groups possess appropriate protecting groups, or unprotected form in which neither amino nor carboxy groups possess an appropriate protecting group. Numerous reactions for the formation and removal of such protecting groups are described in a number of standard works including, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d , Plenum Press (London and New York, 1973); Green, T. H., xe2x80x9cProtective Groups in organic Synthesisxe2x80x9d , Wiley (New York, 1981); and xe2x80x9cThe Peptidesxe2x80x9d Vol. I, Schrxc3x6der and Lxc3xcbke, Academic Press (London and New York, 1965).
Representative amino protecting groups include, for example, formyl, acetyl, isopropyl, butoxycarbonyl, fluorenylmethoxycarbonyl, carbobenzyloxy, and the like.
Representative carboxy protecting groups include, for example, benzyl ester, methyl ester, ethyl ester, t-butyl ester, p-nitro phenyl ester, and the like.
In addition to protected forms in which both amino and carboxy groups possess appropriate protecting groups, the term xe2x80x9cprotectedxe2x80x9d also refers to those GLP-1 molecules in which the activity of dipeptidyl-peptidase IV is resisted or inhibited [see, e.g., Mentlein, R., et al., Eur. J. Biochem., 214:829-835 (1993)]. In addition to GLP-1(7-36)NH2, molecules which are protected from the activity of DPP IV are preferred, and Gly8-GLP-1(7-36)NH2, Val8-GLP-1(7-37)OH, xcex1-methly-Ala8-GLP-1(7-36)NH2, and Gly8-Gln21-GLP-1(7-37)OH are more preferred.
Derivatives of naturally-occurring GLP-1 molecules are those peptides which are obtained by fragmenting a naturally-occurring sequence, or are synthesized based upon a knowledge of the sequence of the naturally-occurring amino acid sequence of the genetic material (DNA or RNA) which encodes this sequence. The term xe2x80x9cderivativesxe2x80x9d also includes chemical modification of natural or unnatural GLP-1 molecules. Processes for preparing these derivatives are well known to organic and peptide chemists of ordinary skill (see, e.g., WO 91/11457, supra).
GLP-1 molecules of the present invention also include analogs of GLP-1 (7-36) NH2 and GLP-1 (7-37) in which one or more amino acids which are not present in the original sequence are added or deleted, and derivatives thereof. Specifically, His and desamino-histidine are preferred for R1, so long as the overall isoelectric point of the molecule is in the range of about 6 to 9. Ala, Gly, and Val are preferred at the xe2x80x9cXxe2x80x9d position, so long as the overall isoelectric point of the mnolecule is in the range of about 6 to 9. Likewise, Glu, and Gln are preferred at the xe2x80x9cYxe2x80x9d position, so long as the overall isoelectric point of the molecule is in the range of about 6 to 9. Also, Glu, and Gln are preferred at the xe2x80x9cZxe2x80x9d position, so long as the overall isoelectric point of the molecule is in the range of about 6 to 9. Finally, Gly-OH is preferred for R2, so long as the overall isoelectric point of the molecule is in the range of about 6 to 9.
Furthermore, the present invention includes a salt form of a GLP-1 molecule. A GLP-1 of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and, especially, hydrochloric acid.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like. The salt forms are particularly preferred.
Of course, when the compounds of this invention are used for pharmacotherapeutic purposes, those compounds may also be in the form of a salt, but the salt must be pharmaceutically acceptable.
Thus, GLP-1 molecules of the present invention include inter alia, those GLP-1 molecules which functionally demonstrate insulinotropic activity. The term xe2x80x9cinsulinotropic activityxe2x80x9d relates to the ability of a substance to stimulate, or cause the stimulation of, the synthesis or expression of the hormone insulin.
The insulinotropic property of a compound may be determined by providing that compound to animal cells, or injecting that compound into animals and monitoring the release of immunoreactive insulin (IRI) into the media or circulatory system of the animal, respectively. The presence of IRI is detected through the use of a radioimmunoassay which can specifically detect insulin.
Although any radioimmunoassay capable of detecting the presence of IRI may be employed, it is preferable to use a modification of the assay method of Albano, J. D. M., et al., Acta Endocrinol., 70:487-509 (1972). In this modification, a phosphate/albumin buffer with a pH of 7.4 is employed. The incubation is prepared with the consecutive addition of 500 xcexcl of phosphate buffer, 50 xcexcl of perfusate sample or rat insulin standard in perfusate, 100 xcexcl of anti-insulin antiserum (Wellcome Laboratories; 1:40,000 dilution), and 100 xcexcl of [125I) insulin, giving a total volume of 750 xcexcl in a 10xc3x9775 mm disposable glass tube. After incubation for 2-3 days at 4xc2x0 C., free insulin is separated from antibody-bound insulin by charcoal separation. The assay sensitivity is 1-2 uU/mL. In order to measure the release of IRI into the cell culture medium of cells grown in tissue culture, one preferably incorporates radioactive label into proinsulin. Although any radioactive label capable of labelling a polypeptide can be used, it is preferable to use 3H leucine in order to obtain labelled proinsulin. Labelling can be done for any period of time sufficient to permit the formation of a detectably labelled pool of proinsulin molecules; however, it is preferable to incubate cells in the presence of radioactive label for a 60 minute time period.
Although many cell lines capable of expressing insulin can be used for determining whether a compound has an insulinotropic effect, it is preferable to use rat insulinoma cells, and especially RIN-38 rat insulinoma cells. Such cells can be grown in any suitable medium; however, it is preferable to use DME medium containing 0.1% BSA and 25 mM glucose.
The insulinotropic property of a compound may also be determined by pancreatic infusion. The in situ isolated perfused rat pancreas preparation is a modification of the method of Penhos, J. C., et al., Diabetes, 18:733-738 (1969). Fasted male Charles River strain albino rats, weighing 350-600 g, are anesthetized with an intraperitoneal injection of Amytal Sodium (Eli Lilly and Co.: 160 ng/kg). Renal, adrenal, gastric, and lower colonic blood vessels are ligated. The entire intestine is resected except for about four cm of duodenum and the descending colon and rectum. Therefore, only a small part of the intestine is perfused, minimizing possible interference by enteric substances with glucagon-like immunoreactivity. The perfusate is a modified Krebs-Ringer bicarbonate buffer with 4% dextran T70 and 0.2% bovine serum albumin (fraction V), and is bubbled with 95% O2 and 5% CO2. A nonpulsatile flow, 4-channel roller bearing pump (Buchler polystatic, Buchler Instruments Division, Nuclear-Chicago Corp.) is used, and a switch from one perfusate source to another is accomplished by switching a 3-way stopcock. The manner in which perfusion is performed, monitored, and analyzed follow the method of Weir, G. C., et al., J. Clin. Inestigat. 54:1403-1412 (1974), which is hereby incorporated by reference.
The GLP-1 molecules of the present invention are required to possess a histidine functionality at the amino terminus. GLP-1 molecules of the present invention may also possess a modified histidine functionality in lieu of the required histidine functionality.
The term xe2x80x9cmodified histidinexe2x80x9d refers to a histidine functionality which has been chemically or biologically altered or an altered histidine functionality which has been synthesized de novo, but which retains its metal binding properties.
Numerous such modified histidine functionalities and their preparation are known in the art and include, for example, D-histidine (WO 91/11457), desamino-histidine (WO 92/18531), 2-amino-histidine [Levine-Pinto, H., et al., Biochem. Biophys. Res. Commun., 103(4):1121-1130 (1981)], xcex2-hydroxy-L-histidine [Owa, T, et al., Chemistry Letters, pp. 1873-1874 (1988)], L-homohistidine [Altman, J., et al., Synthetic Commun., 19(11and12) :2069-2076 (1989)], xcex1-fluoromethyl-histidine (U.S. Pat. No. 4,347,374), and xcex1-methylhistidine [O""Donnell, M. J., Synthetic Commun., 19(7and8):1157-1165 (1989)].
The GLP-1 molecules of the present invention further are required to have an isoelectric point in the range from about 6.0 to about 9.0. Numerous GLP-1 molecules having an isoelectric point in this range have been disclosed and include, for example:
GLP-1 (7-36)NH2
Gly8-GLP-1 (7-36)NH2
Gln9-GLP-1 (7-37)
D-Gln9-GLP-1 (7-37)
acetyl-Lys9-GLP-1 (7-37)
Thr9-GLP-1 (7-37)
D-Thr9-GLP-1 (7-37)
Asn9-GLP-1 (7-37)
D-Asn9-GLP-1 (7-37)
Ser22-Arg23-Arg24-Gln26-GLP-l (7-37)
Thr16-Lys18-GLP-1 (7-37)
Lys18-GLP-1 (7-37)
Arg23-GLP-1 (7-37)
Arg24-GLP-1 (7-37), and the like (see, e.g.,
WO 91/11457, supra). In addition, GLP-1 molecules of the present invention, when possessing each of the above-referenced modified histidine functionalities in lieu of the histidine functionality, have isoelectric points which fall within the above-defined range. Methods for calculating or experimentally determining the isoelectric point of other GLP-1 molecules are known to one of ordinary skill in the art.
Methods for preparing the GLP-1 molecules of the present invention also are well known to an ordinarily skilled peptide chemist.
In one method, GLP-1 molecules are prepared by the well-known solid phase peptide synthetic schemes described by Merrifield, J. M., Chem. Soc., 85:2149 (1962), and Stewart and Young, Solid Phase Peptide Synthesis, pp. 24-66, Freeman (San Francisco, 1969). However, it also is possible to obtain fragments of the proglucagon polypeptide or of GLP-1 (1-37) by fragmenting the naturally-occurring amino acid sequence using, for example, a proteolytic enzyme. Further, it is possible to obtain the desired fragments of the proglucagon peptide or of GLP-1 (1-37) through the use of recombinant DNA technology as disclosed by Maniatis, T., et al., Molecular Biology: A Laboratory Manual, CSH (Cold Spring Harbor, 1982).
Likewise, the state of the art in molecular biology provides the ordinarily skilled artisan another means by which compounds of the present invention can be obtained. Although it may be produced by solid phase peptide synthesis or recombinant methods, recombinant methods may be preferable because higher yields are possible. The basic steps in recombinant production are:
a) isolating a natural DNA sequence encoding a GLP-1 molecule or constructing a synthetic or semi-synthetic DNA coding sequence for a GLP-1 molecule,
b) placing the coding sequence into an expression vector in a manner suitable for expressing proteins either alone or as a fusion proteins,
c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector,
d) culturing the transformed host cell under conditions that will permit expression of a GLP-1 molecule, and
e) recovering and purifying the recombinantly produced GLP-1 molecule.
As previously stated, the coding sequences may be wholly synthetic or the result of modifications to the larger, native glucagon-encoding DNA. A DNA sequence that encodes preproglucagon is presented in Lund, et al., Proc. Natl. Acad. Sci. U.S.A. 79:345-349 (1982) and may be used as starting material in the semisynthetic production of the compounds of the present invention by altering the native sequence to achieve the desired results.
Synthetic genes, the in vitro or in vivo transcription and translation of which results in the production of a GLP-1 molecule, may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed, all of which encode GLP-1 molecules.
The methodology of synthetic gene construction is well known in the art. See Brown, et al. (1979) Methods in Enzymology, Academic Press, N.Y., Vol. 68, pgs. 109-151. DNA sequences that encode a GLP-1 molecule can be designed based on the amino acid sequences herein disclosed. Once designed, the sequence itself may be generated using conventional DNA synthesizing apparatus such as the Model 380A or 380B DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404).
To effect expression of a GLP-1 molecule, one inserts the engineered synthetic DNA sequence in any one of many appropriate recombinant DNA expression vectors through the use of appropriate restriction endonucleases. See generally Maniatis et al. (1989) Molecular Cloning; A Laboratory Manual, Cold Springs Harbor Laboratory Press, N.Y., Vol. 1-3. Restriction endonuclease cleavage sites are engineered into either end of the GLP-1 molecule-encoding DNA to facilitate isolation from, and integration into, known amplification and expression vectors. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed. The choice of restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in-frame reading and expression of the protein of interest. The coding sequence must be positioned so as to be in proper reading frame with the promoter and ribosome binding site of the expression vector, both of which are functional in the host cell in which the protein is to be expressed.
To achieve efficient transcription of the synthetic gene, it must be operably associated with a promoter-operator region. Therefore, the promoter-operator region of the synthetic gene is placed in the same sequential orientation with respect to the ATG start codon of the synthetic gene.
A variety of expression vectors useful for transforming prokaryotic and eukaryotic cells are well known in the art. See The Promega Biological Research Products Catalogue (1992) (Promega Corp., 2800 Woods Hollow Road, Madison, Wis., 53711-5399); and The Stratagene Cloning Systems Catalogue (1992) (Stratagene Corp., 11011 North Torrey Pines Road, La Jolla, Calif, 92037). Also, U.S. Pat. No. 4,710,473 describes circular DNA plasmid transformation vectors useful for expression of exogenous genes in E. coli at high levels. These plasmids are useful as transformation vectors in recombinant DNA procedures and
(a) confer on the plasmid the capacity for autonomous replication in a host cell;
(b) control autonomous plasmid replication in relation to the temperature at which host cell cultures are maintained;
(c) stabilize maintenance of the plasmid in host cell populations;
(d) direct synthesis of a protein prod. indicative of plasmid maintenance in a host cell population;
(e) provide in series restriction endonuclease recognition sites unique to the plasmid; and
(f) terminate mRNA transcription.
These circular DNA plasmids are useful as vectors in recombinant DNA procedures for securing high levels of expression of exogenous genes.
Having constructed an expression vector for a GLP-1 molecule, the next step is to place the vector into a suitable cell and thereby construct a recombinant host cell useful for expressing the polypeptide. Techniques for transforming cells with recombinant DNA vectors are well known in the art and may be found in such general references as Maniatis, et al. supra. Host cells made be constructed from either eukaryotic or prokaryotic cells.
Prokaryotic host cells generally produce the protein at higher rates and are easier to culture. Proteins which are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. Such protein aggregates typically must be solubilized, denatured and refolded using techniques well known in the art. See Kreuger, et al. (1990) in Protein Folding, Gierasch and King, eds., pgs 136-142, American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C.; and U.S. Pat. No. 4,923,967.
Once the desired GLP-1 molecule is prepared, providing it has an isoelectric point in the range from about 6.0 to about 9.0, complexes of the present invention are prepared by complexing a desired GLP-1 molecule with a divalent metal cation via well known methods in the art. Such metal cations include, for example, Zn++, Mn++, Fe++, Co++, Cd++, Ni++, and the like. Of the metal cations, Zn++ is preferred.
Generally, a desired GLP-1 molecule, having the required isoelectric point, is combined with a mixture of an appropriate buffer and an appropriate form of a metal cation.
Appropriate buffers are those which will maintain the mixture at a pH range from about 6.0 to about 9.0, but which will not interfere with the reaction. Preferred buffers include Goode""s buffers, particularly HEPES, and Tris and Tris acetate.
Appropriate forms of metal cations are any form of a divalent metal cation which is available to form a complex with a GLP-1 molecule of the present invention. Preferably, a divalent metal cationic salt such as zinc chloride is provided in excess to provide a molar ratio of up to about 50 molecules of a divalent metal cation for each molecule of GLP-1 substrate.
The temperature employed in this step is that which is sufficient to effect completion of the reaction. Typically, the reaction is run at ambient temperature.
The product of the present reaction, a crystalline or amorphous suspension, is isolated and purified using standard techniques.
The present invention also provides pharmaceutical compositions comprising a compound of the present invention in combination with a pharmaceutically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art, and are administered individually or in combination with other therapeutic agents, preferably via parenteral routes. Especially preferred routes include intramuscular and subcutaneous administration.
Parenteral daily dosages, preferably a single, daily dose, are in the range from about 1 pg/kg to about 1,000 xcexcg/kg of body weight, although lower or higher dosages may be administered. The required dosage will depend upon the severity of the condition of the patient and upon such criteria as the patient""s height, weight, sex, age, and medical history.
In making the compositions of the present invention, the active ingredient, which comprises at least one compound of the present invention, is usually mixed with an excipient or diluted by an excipient. When an excipient is used as a diluent, it may be a solid, semi-solid, or liquid material which acts as a vehicle, carrier, or medium for the active ingredient.
In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to particle size of less than about 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g., about 40 mesh.
Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, and mannitol. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art.
The compositions are preferably formulated in a unit dosage form with each dosage normally containing from about 50 xcexcg to about 100 mg, more usually from about 1 mg to about 10 mg of the active ingredient. The term xe2x80x9cunit dosage formxe2x80x9d refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient.
For the purpose of parenteral administration, compositions containing a compound of the present invention preferably are combined with distilled water and the pH is adjusted to about 6.0 to about 9.0.
Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved by the use of polymers to complex or absorb a compound of the present invention. The controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylenevinyl acetate, methylcellulose, carboxymethylcellulose, and protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
Another possible method to control the duration of action by controlled release preparations is to incorporate a compound of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers.
Alternatively, instead of incorporating a compound into these polymeric particles, it is possible to entrap a compound of the present invention in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or in macroemulsions. Such teachings are disclosed in Remington""s Pharmaceutical Sciences (1980).
The compounds of the present invention have insulinotropic activity. Thus, another aspect of the present invention provides a method for enhancing the expression of insulin comprising providing to a mammalian pancreatic B-type islet cell an effective amount of a compound of the present invention.
Similarly, the present invention provides a method for treating maturity onset diabetes mellitus in a mammal, preferably a human, in need of such treatment comprising administering an effective amount of a compound or composition of the present invention, to such a mammal.

Glucagon-like insulinotropic peptides, compositions and methods


Electrochromic assemblies having a layer structure, characterized in that two layers each contain an electrically conductive, electrochromic polydioxythiophene selected from the same group of substances.

The present invention relates to electrochromic assemblies having controllable light transmittance, its production and its use.
The transparency of windows of vehicles in respect of electromagnetic radiation has hitherto not been able to be regulated. Phototropic glasses have hitherto been used only as glass in spectacles and have only a relatively small change in the transmission. Windows of buildings have hitherto been darkened by means of curtains, shutters, roller blinds or other movable mechanical elements. Electrochromic devices can thus be employed in a wide variety of ways. A brief overview of examples is as follows:
1. Vehicle glazing (windows or sunroofs of automobiles)
An electrochromic device is suitable as protection against sun or dazzling in motor vehicles. Front, side and rear windows or glass roofs can be included. The degree of darkening can be matched zone wise and steplessly to the needs of the driver depending on the position of the sun and the immediate driving situation. Integration into a computer-controlled regulating system is possible. A combination of an active element with a laminated glass unit is likewise possible, for example application of a film system to the safety glass.
The transmittance of the windows can be controlled manually or automatically, which can be used for effective protection against dazzling during night driving, automatic adjustment of the level of brightness on driving into and out of tunnels and multistorey car parks and for protection against forcible entry and theft when the vehicle is parked by preventing a view into the interior of the vehicle. Excessive heating of the interior in summer, particularly when the vehicle is parked can be prevented (cf. EP-A 0,272,428).
2. Glazing of buildings (electrochromic window)
In buildings, electrochromic assemblies are suitable for darkening side windows and skylights of buildings, living areas, workrooms or greenhouses as controllable sun protection (visible spectral region) and heat protection (IR region) and also for protection of the eyes (visible spectral region). For protection against break-ins, glazing of bank counters or shop windows can be darkened on the press of a button. Glass doors can automatically be made visible on the approach of persons in order to avoid injury. The ability to generate virtually all colours also makes it possible to incorporate the glazing architecturally into the facade of a building. The energy consumption for controlling the transparency of a large area of window is low, particularly when the memory effect of the system can be exploited and energy is only consumed in the switching phase. A combination with heat-protection glazing (K glass) is very well suited to achieving dynamic control of the sunlight shining through a window (xe2x80x9csmart windowxe2x80x9d). Thus, an electrochromic system can contribute to regulating and limiting the energy required for air conditioning of buildings.
The power supply to the system can also be achieved by means of solar modules. A light-sensitive sensor can determine the intensity of the sunlight and thus control the light transmittance.
3. Display elements
The ability to produce attractive colours and display any desired contours, e.g. letters, numbers, signs and symbols (able to be produced by appropriate structuring techniques) on a large area provides advertizing with an interesting medium. Decorative and informative effects are readily possible.
Apart from the possibility of locating the system between panes of glass, there is also the alternative of using two or even only one transparent plastic film as support. This makes it possible to achieve placard-like advertizing materials with changeable information.
Electrochromic devices can be used for small display elements such as faces of watches and clocks or measuring instruments, displays for a wide variety of applications and for large display elements such as traffic signs, advertizing columns, information displays at railway stations and airports or for providing parking directions. Use as variable delineation system (marking of boundaries etc. on playing areas) in sports halls is likewise possible.
They can be used wherever information is to be made visible.
4. Optics
In optics, electrochromic systems can be used either in combination with glasses, lenses and filters of other optical instruments as well as sole active components. Use as fade-over protection for optical detection systems is likewise possible. The system is likewise suitable as a controllable filter system in photographic processes.
5. Mirrors
An electrochromic device can also be used as a dimmable mirror, e.g. in an automobile as external or rear-view mirror, which can be darkened by application of an electric potential and thus prevents dazzling by the headlights of other vehicles (cf., for example, U.S. Pat. No. 3,280,702, U.S. Pat. No. 4,902,108 (Gentex), EP-A 0,435,689, U.S. Pat. No. 5,140,455). A disadvantage of systems of the prior art (solution systems) is the colour in homogeneity after prolonged operation (segregation), particularly in the case of large mirrors (e.g. mirrors of goods vehicles). Increasing the viscosity of the solution system by addition of polymeric thickeners has been described (e.g. U.S. Pat. No. 4,902,108).
6. EMI shielding
An electrochromic device can also be used as a variable filter element for modulating electromagnetic radiation in certain wavelength ranges.
Electrochromic devices usually comprise a pair of glass or plastic plates of which one is mirrored in the case of a mirror. One side of each of these plates is coated with a translucent electrically conductive layer, e.g. indium-tin oxide (ITO). These plates are used to construct a sill by fixing them with their conductively coated sides facing one another. The cell between the plates contains the electrochromic system and is closed tightly. The two plates can be separably connected to a power source and controlled via the conductive layer.
In the electrochromic solution systems known from the above-cited prior art, pairs of redox substances which after reduction or oxidation form coloured, positively or negatively charged free radicals which are chemically reactive are present in a solvent. Examples are the viologen systems which have been known for a long time.
As the pair of redox substances, use is made of one reducible and one oxidizable substance. Both are colourless or have only a slight colour. Under the action of an electric potential, one substance is reduced and the other is oxidized, with at least one becoming coloured. After the potential is switched off, the two original redox substances are formed again, with decolouration or lightening of the colour occurring.
It is known from U.S. Pat. No. 4,902,108 that pairs of redox substances in which the reducible substance has at least two chemically reversible reduction waves in the cyclic voltammogram and the oxidizable substance correspondingly has at least two chemically reversible oxidation waves are suitable. Systems of this type are suitable mainly for dimmable rear view mirrors of automobiles. Since these are solution systems, they are normally not suitable for use in electrochromic windows.
Also known are systems in which the actual electrochromic redox pair is dispersed in a polymer matrix (see, for example, WO-A 96/03475). The undesirable effect of segregation is suppressed in this way.
Combinations of inorganic electrochromic components such as WO3, NiO or IrO2 are likewise known and are possibilities as components in an electrochromic window (see, for example, U.S. Pat. No. 5,657,149, Electronique International No. 276, 16 (1997)).
These inorganic electrochromic components can be applied to the conductive substrate only by vapour deposition, sputtering or by a sol-gel technique. As a result, systems of this type are very expensive to produce. Efforts to replace one inorganic component by an organic polymer component have resulted in, for example, electrochromic systems based on the electrically conductive polymer polyaniline (PANI) and WO3 as complementary electrochromic materials becoming known (see, for example, B. P. Jelle, G. Hagen, J. Electrochem. Soc., Vol. 140, No. 12,3560 (1993)). An attempt has also been made to use systems without an inorganic component in which the ITO or SnO2, layer (counterelectrode) is supposed to serve as complementary electrochromic component or ion-storage layer to substituted poly(3,4-alkylenedioxythiophenes) (U.S. Pat. No. 5,187,608).
However, it is found that such electrochromic assemblies are not able to ensure a sufficient number of switching cycles without a change occurring in the properties of the device.
The present invention provides an electrochromic assembly having a layer structure, characterized in that two layers contain an electrically conductive, electrochromic polydioxythiophene. Here, one layer acts as a colour-imparting electrochromic layer while the second layer acts as an ion-storage layer. In the layer structure, the ion-storage layer comprising a polydioxythiophene derivative can have a lower thickness than the colour-imparting electrochromic layer. In addition, the polydioxythiophenes in the colour-imparting layer and the ion-storage layer can be two different derivatives from this group of substances.
The ion-storage layer in the assembly of the invention preferably comprises the same polydioxythiophene as the colour-imparting component, but the ion-storage layer preferably has a lower thickness. The ion-storage layers can include an Li salt when they are produced or else can be loaded electrochemically with Li ions afterwards.
The polydioxythiophenes are cationically charged and comprise structural units of the formula (I)
where
A1 and A2 each represent, independently of one another, substituted or unsubstituted (C1-C4)-alkyl or together form substituted or unsubstituted (C1-C4)-alkylene,
and
n represents an integer from 2 to 10,000, preferably from 5 to 5,000, in the presence of polyanions.
Preferred cationic polydioxythiophenes comprise structural units of the formula (Ia) or (Ib)
where
R1 and R2 represent, independently of one another, hydrogen, substituted or unsubstituted (C1-C18)-alkyl, preferably (C1-C10)-, in particular (C1-C6)-alkyl, (C2-C12)-alkenyl, preferably (C2-C8)-alkenyl, (C3-C7)-cycloalkyl, preferably cyclopentyl or cyclohexyl, (C7-C15)-aralkyl, preferably phenyl-(C1-C4)-alkyl, (C6-C10)-aryl, preferably phenyl or naphthyl, (C1-C18)-alkyloxy, preferably (C1-C10)-alkyloxy, for example methoxy, ethoxy, n- or iso-propoxy, or (C2-C18)-alkyloxy ester and
R3, R4 represent, independently of one another, hydrogen, but not both at the same time, or (C1-C18)-alkyl, preferably (C1-C10)-, in particular (C1-C6)-alkyl, (C2-C12)-alkenyl, preferably (C2-C8)-alkenyl, (C3-C7)-cycloalkyl, preferably cyclopentyl or cyclohexyl, (C7-C15)-aralkyl, preferably phenyl-(C1-C4)-alkyl, (C6-C10)-aryl, preferably phenyl or naphthyl, (C1-C18)-alkyloxy, preferably (C1-C10)-alkyloxy, for example methoxy, ethoxy, n- or iso-propoxy, or (C2-C18)-alkyloxy ester each of which are substituted by at least one sulphonate group
n represents a number from 2 to 10 000, preferably from 5 to 5,000.
Very particularly preferably, the electrochromic device of the invention contains at least one electrically conductive, electrochromic cationic or uncharged polydioxythiophene of the formulae (I a-1) and/or (I b-1)
where
R3 is as defined above,
n represents an integer from 2 to 10,000, preferably from 5 to 5,000.
The polyanions are the anions of polymeric carboxylic acids such as polyacrylic acids, polymethacrylic acids or polymaleic acids or of polymeric sulphonic acids such as polystyrenesulphonic acids and polyvinylsulphonic acids. These polycarboxylic and polysulphonic acids can also be copolymers of vinylcarboxylic and vinylsulphonic acids with other polymerizable monomers such as acrylic esters and styrene.
The anion of polystyrenesulphonic acid is particularly preferred as counterion.
The molecular weight of the polyacids providing the polyanions is preferably from 1000 to 2,000,000, particularly preferably from 2000 to 500,000. The polyacids or their alkali metal salts are commercially available, e.g. polystyrenesulphonic acids and polyacrylic acids, or else can be prepared by known methods (see, for example, Houben-Weyl, Methoden der organischen Chemie, vol. E 20 Makromolekulare Stoffe, part 2, (1987), p. 1141 ff.).
In place of the free polyacids required for the formation of dispersions of polydioxythiophenes and polyanions, it is also possible to use mixtures of alkali metal salts of the polyacids and corresponding amounts of monoacids.
In the case of the formula (Ib 1), the polydioxythiophenes bear positive and negative charges in the structural unit. The preparation of the polydioxythiophenes is described, for example, in EP-A 0,440,957 (=U.S. Pat. No. 5,300,575).
The polydioxythiophenes are obtained by oxidative polymerization. As a result they acquire positive charges which are not shown in the formulae, since their number and position cannot be unambiguously determined.
The present invention accordingly provides an electrochromic device containing electrically conductive poly(3,4-ethylenedioxythiophene) derivatives as cathodically colouring electrochromic polymers and ion-storage layers for Li ions, each of which are selected from the same group of poly(3,4-ethylenedioxythiophene) derivatives. A gel electrolyte comprising a crosslinked or uncrosslinked polymer, an Li salt and a certain amount of a solvent is located between the electrochromic polymer layer and the polymeric ion-storage layer. The schematic structure is shown in FIG. 1, principle I).
Legend for FIG. 1:
1,2: substrate
3,4: electrically conductive coating, of which one can act as a mirror
5: electrochromic polymer, e.g. PEDT/PSS
6: ion-storage layer, e.g. PEDT/PSS having a lower thickness than in 5.
7: gel electrolyte (crosslinked or uncrosslinked)
8,9: fine metal grid (optional)
The electrochromic polymer layer is transparent in the doped state. This can be converted into a coloured form by uptake of electrons (reduction) at the cathode with an increase in the absorbance in the visible region of the spectrum. The oxidation which occurs on the opposite side (anode) is associated with an exchange reaction of the ion-storage layer with Li ions. However, due to the preferred lower layer thickness this reaction barely contributes to the generation of colour, so that it does not interfere. Owing to the lower thickness of the ion-storage layer, the decoloured state shows sufficient transparency.
The present invention accordingly provides an electrochromic solid-state system containing at least two redox-active electrically conductive polymers selected from the group consisting of poly(3,4-ethylenedioxythiophene) derivatives which can, to enable them to be processed from solution, have been admixed with polystyrenesulphonate or bear a solubilizing sulphonate group in a side chain. This polymer layer is preferably applied from aqueous solution, in which case the solvent is evaporated to leave the solid, dry polymer film on the substrate. However, it should also be possible to apply it by screen printing. As substrates, preference is given to using an electrically conductive, transparent glass or film system where a layer of indium-tin oxide (ITO), fluorine-doped tin oxide (FTO), K-Glas), undoped tin oxide or a layer of finely divided silver serves as electrode. It is also possible for one electrode side to consist of a metal layer e.g. Al, Cu, Pd) which is no longer transparent (for use in a mirror). The gel electrolyte contains at least one polymer (e.g. polyethylene oxide, PMMA), at least one Li salt (e.g. Li triflate, Li perchlorate), at least one solvent (e.g. propylene carbonate).
The present invention provides for the use of the electrochromic device of the invention in the glazing of buildings or architectural glazing or sunroof in vehicles and also as display element, as electrochromic mirror (e.g. automatically dimming rear view mirror in automobiles) and in various optical elements.
For use as a mirror, one of the two electrodes can consist of a vapour-deposited or electrochemically deposited metal layer, e.g. aluminium, silver, copper, platinum, palladium or rhodium.
The present invention also provides an electrochromic system in which the colour-producing electrochromic polymer compound functions simultaneously as its own electrode, as a result of which only a conductive coating of ITO, fluorine-doped tin oxide or a metal is necessary. (see FIG. 1, principle II)).
Legend for FIG. 1, principle II:
1,2: substrate
4: electrically conductive coating which can also act as a mirror
5: electrochromic polymer
6: ion-storage layer
7: gel electrolyte (crosslinked or uncrosslinked)
8,9: fine metal grid (optional)
The electrochromic assembly of the invention is particularly notable for the fact that a combination with a heat-protection glass (commercially available for architectural glazing purposes) explicitly as a positive feature of the assembly is possible for saving energy in the case of brightly sunlit rooms. Further explicit electrodes of another material are thus unnecessary, since the heat-protection layer limits the transmission of IR radiation and at the same time, due to its electric conductivity, assumes the electrode function in the electrochromic assembly.
The electrochromic assembly of the invention is also notable for the fact that the electrochromic layer can also absorb IR radiation in certain ranges and can thus limit the passage of heat through the pane.
The electrochromic layer structure of the invention is suitable as a constituent of an electrochromic device. In an electrochromic device, the electrochromic assembly of the invention serves as a medium having variable transmission, i.e. the light transmittance of the system alters under the action of an electric potential as a result of it changing from a colourless to a coloured state. The present invention therefore also provides electrochrornic devices containing an electrochromic assembly according to the invention. Applications of this electrochromic device are in architectural glazing and in vehicles, e.g. as window, automobile sunroof, rear view mirror in an automobile, display or as an optical element or as constituent of information display units such as instrument displays in vehicles of all types.
If the electrochromic device is a electrochromic display device, at least one of the two conductive layers or both is/are divided into electrically separate segments which are individually connected to a power source.
However, it is also possible for only one of the two plates to have a conductive coating and to be divided into segments. The segments can be separated, for example, by mechanical removal of the conductive layer, e.g. by scoring, scratching, scraping or milling, or by chemical means, for example by etching using, for example, a hydrochloric acid solution of FeCl2 or SnCl2. The location of this removal of the conductive layer can be controlled by means of masks, e.g. masks of photoresist. However, the electrically separate segments can also be produced by targeted, e.g. by means of masks, application, e.g. by sputtering or printing, of the conductive layer. The segments are connected to a power source by means of, for example, fine strips of conductive material so that the segment is electrically connected to a contact at the edge of the electrochromic device. These fine contact strips can consist of the same material as the conductive layer itself and can be produced together with it, for example when it is divided into segments. However, they can also, e.g. to improve the conductivity, consist of another material such as fine metallic conductors, for example of copper or silver. A combination of metallic material and the material of the conductive coatirrg is also possible. The metallic conductors can, for example, either be applied in fine wire form, e.g. adhesively bonded on, or be printed on. All these above-described techniques are generally known from the production of liquid-crystal displays (LCDs).
In the case of displays, the displays produced according to the invention can be viewed in transmitted light or in reflected light by means of mirroring.
If the electrochromic device is an electrochromic window, a fine metal grid can be vapour-deposited on one or both electrodes. This improves the surface conductivity of the substrates and is advantageous in the case of large areas in order to achieve uniform colouring.
The electrochromic assembly of the invention preferably contains at least one transparent electrically conductive coating comprising indium-tin oxide (In2O3: SnO2 (ITO)), tin oxide (SnO2), fluorine-doped tin oxide (SnO2: F; FTO or xe2x80x9cK-glassxe2x80x9d, xe2x80x9cheat-protection glassxe2x80x9d), antimony-doped tin oxide, antimony-doped tin oxide, aluminum-doped zinc oxide or a transparent metal film which is sufficiently thin, e.g. silver coating (heat-protection glass), on a substrate (glass or plastic).
Other conductive polymers such as substituted or unsubstituted polythienyls, polypyrroles, polyanilines, polyactetylene or polythiophenes can also be used.
In the assembly of the invention, the actual electrochromic polymer is advantageously also used as its own conductive electrode material in place of one of the abovementioned conductive coatings.
Very particular preference is given to using indium-tin oxide (In2O3:SnO2 (ITO)), tin oxide (SnO2), fluorine-doped tin oxide (SnO2: F; FTO, xe2x80x9cK-glassxe2x80x9d, xe2x80x9cheat-protection glassxe2x80x9d) or a transparent silver coating which is sufficiently thin (heat-protection glass).
If one of the plates is mirrored, this conductive layer can also be utilized. Particular preference is here given to using silver, aluminium, copper, platinum, palladium and rhodium.
The electrochromic assembly of the invention preferably contains a transparent gel electrolyte comprising the following components:
polymer (crosslinked or uncrosslinked)
Li salt
solvent or solvent mixture
Preferred polymers are polymethyl methacrylate (PMMA), polyethylene oxide(PEO), polyacrylonitrile (PAN), poly(N,N-dimethylacrylamide), poly(2-(2-methoxyethoxy)-ethoxy) phosphazene, poly(oxymethylene-oligo(oxyethylene)), polyethylene glycols (PEGs), polypropylene glycols (PPG) or polymers based on polyepichlorohydrin or polyethers and also mixtures thereof. Copolymers such as ethylene oxide-propylene oxide (EO/PO) copolymers or oxymethylene-bridged polyethylene oxides are also suitable.
Particular preference is given to using polyethers and polyethylene oxides.
Particular preference is also given to photocrosslinkable polymer systems based on acrylates, e.g. polyethylene glycol 400 diacrylate, polyethylene glycol 400 dimethacrylate, polyethylene glycol 600 diacrylate, polyethylene glycol 600 dimethacrylate, polyethylene glycol methacrylate, tripropylene glycol diacrylate, tripropylene glycol monomethyl ether acrylate, trimethylolpropane triacrylate, ethylene glycol dimethacrylate hydroxyethyl methacrylate (HEMA), hexanediol diacrylate, dianol diacrylate, tetraethylene glycol diacrylate, pentaerythritol triacrylate, pentaerythritol tetracrylate, butyl methacrylate. The photocrosslinkable polymer systems should still be able to be cured in the presence of the solvent used and the Li salt with the aid of light activation by means of a customary photoinitiator such as (copyright)Darocure 1173, 1116 or (copyright)Irgacure 184 (E. Merck KGaA, Darmstadt) even between thick glass plates which are provided with a transparent electrically conductive coating. Illumination is carried out after filling the cell by irradiation with a suitable lamp (e.g. UV lamps such as Hg or Xe lamps). Curing of polymer systems by electron beam curing is likewise possible for the systems mentioned.
Very particular preference is also given to polymer systems which can be crosslinked thermally and catalytically via isocyanate groups with OH-functional polyether compounds, e.g. polyether polyols, to form polyurethanes. Polyurethanes having different soft segments, e.g. polytetramethylene glycol or polypropylene glycol, are also suitable.
Very particular preference is also given to modified siloxanes derived from, for example, gamma-glycidylpropyltrimethoxysilane. Variants modified by means of propylene oxide, for example, are also possible.
The gel electrolytes can also contain organic and/or inorganic fillers or additives. Here, the customary additive such as heat stabilizers, optical brighteners, flame retardants, flow improvers, fire retardants, dyes, pigments, fillers or reinforcing materials, finely divided minerals, fibres, chalk, quartz flour, glass, aluminium oxide, aluminium chloride and carbon fibres can be added in customary amounts. The function of a spacer can be performed, for example, by glass spheres, polymer particles, silica gel or sand grains having a defined size, should this be necessary.
Preferred Li salts are LiClO4, LiCF3SO3, LiN(SO2CF3)2, LiCl, LiPF6.
Very particular preference is here given to LiClO4, LiCF3SO3 and LiN(SO2CF3)2.
Particularly preferred solvents are propylene carbonate, ethylene carbonate, acetonitrile and xcex3-butyrolactone and also mixtures thereof.
Very particular preference is given tousling propylene carbonate and ethylene carbonate.
Substrates used in the electrochromic assembly of the invention are glass or various types of plastic.
Preference is given to transparent substrates of any type.
Apart from glass, specifically heat-protection glass when used as electrochromic window (in thicknesses of 10 xcexcm in the case of xe2x80x9cflexible glass, thin glassxe2x80x9d to 3 cm), particularly preferred materials are polyesters (e.g. polyethylene terephthalate (PET) or polyethylene naphthalate (PEN)), various types of polycarbonate (e.g. (copyright)Makrolon, APEC-HT), polysulphones, polyimides and polycycloolefins. The polymeric substrate can be used as flexible film or as a thick plate. The substrate can also be curved so that the assembly matches the shape of the material underneath. A flexible plastic substrate can also, after construction of the overall electrochromic system, be laminated or adhesively bonded onto various materials, e.g. curved glass.
The plastic substrates can additionally be provided with barrier layers against water and oxygen.
Preference is here given to TiOx, SiOx on polyester, e.g. polyethylene terephthalate or fluorinated polymers and possible combinations thereof and also barrier layers based on inorganic-organic hybrid systems.
The electrochromic assembly of the invention can, when configured as a flexible film system, be laminated or adhesively bonded as complete electrochromic composite system onto the safety glass of automobiles. In addition, it can be integrated into the hollow space of a double glazing system in buildings.
The control mechanism of the electrochrornic assembly is based on the reversible electrochemical doping of the electrochromic polymer which results in great colour changes, for example from colourless to blue. The assembly is driven by means of defined voltages.
The reduction and oxidation processes in the electrochromic assembly of the invention generally occur by electron uptake and release at the cathode and anode, respectively, and the potential difference between the electrodes is preferably from 0.1 to 5 V, very particularly preferably from 0.1 to 3 V. After the electric potential is switched off, the previously achieved coloration can be maintained for some time (memory effect) so that permanent coloration can be achieved with minimum energy consumption. Charge equilibration and thus decolouration can be achieved by brief reversal of the polarity.
The electrochromic assembly of the invention can be supplied with power by means of solar modules, even in the case of relatively large areas.
In addition, it was found that the electrochromic polymer film does not have to be produced in-situ on the electrically conductive substrate, but it can also be applied from an ecologically acceptable aqueous solution by means of a casting technique, by spin coating/dip coating, by screen printing or by spraying. This method is particularly suitable for systems having a large area.
To improve wetting of the substrates, it is also possible to add a wetting agent (e.g. Fluortensid)

Electro chromic assembly based on poly (3,4-ethylenedioxythiophene derivatives in the electrochromic layer and the ion-storage layer


The invention concerns an anhydrous solid composition comprising at least a hydrophobic wax, an oil and talcum, having preferably the form of beads with size ranging from 1 to 10000 microns. The beads can contain a cosmetic or pharmaceutical active principle, pigments or an agri-food constituent. The invention also concerns the method for preparing said beads.

This invention concerns a new type of bio-compatible pearl capable of containing various substances, biologically active constituents, cosmetic or pharmaceutical constituents, pigments or load products.
The use of active principles, or any compound or substance, for topical applications with a dermo-pharmaceutical or cosmetic purpose, requires incorporation into a support, referred to as a xe2x80x9cbasexe2x80x9d for cosmetic applications and a xe2x80x9cgalenic supportxe2x80x9d for pharmaceutical applications.
This support has numerous functions:
it must allow practical application at the administration point. In the case of local application with a cosmetic or dermo-pharmaceutical purpose, a fluid with an adapted rheologic behaviour is the form of support the most often chosen.
it must allow the dilution of the active principle or the active compounds to obtain the desired concentration compatible with the effect sought.
it must provide, in the case of cosmetic products, a feeling to the touch as agreeable as possible, and, in this case, the sensorial properties of the support are of primary importance.
The majority of cosmetic or dermo-pharmaceutical bases for topical applications are aqueous compounds, for example gels, lotions and emulsions. These cosmetic products are makeup removers, toning products, slimming gels, after-sun creams or lotions, capillary products, personal care products, tooth pastes and certain makeup products. Dermo-pharmaceutical compounds also use the such supporting bases.
These aqueous bases are today very widely used due to their high water content that gives, on application, a sensation of coolness on the skin and mucous. They leave no residue, do not shine and do not give a greasy aspect or feeling to the skin.
However, these aqueous bases, in particular, those in continuous aqueous phase, present a certain number of inconveniences recalled in patent FR 2 660554. Briefly, we note the drying effect of these bases on the skin, which is badly tolerated with dry or normal skin. The capacity of gels and emulsions to incorporate certain ingredients, such as essential oils or perfumes, without being destabilised, remains weak.
The bases of the emulsion type, containing a greasy phase, present a very creamy consistency. They are very much sought after since they are easily applied with the fingers from ajar or tube container (Patent FR 2734714). The great stability of these emulsions is due to the presence of waxes in the greasy phase whose melting point is generally higher than 80xc2x0 C. Their preparation, at a temperature exceeding the wax melting point, prevents the use of thermo-sensitive active principles.
In addition to the drying effect observed, the aqueous phase is very favourable to the development of micro-organisms (Pharmacie galxc3xa9nicxe2x80x94Bonnes pratiques de fabrication des medicamentsxe2x80x94A. Le Hir, Ed. Masson, page 88). Agents that limit the development of a wide spectrum of microbes must therefore be incorporated into these preparations. These agents, called preservatives, can be toxic for the skin of some people. Thus the use of certain preservatives is today highly regulated or even prohibited for some, for example diisobutyl-phenoxy-ethoxy-ethyl-dimethyl-benzylammonium, (Legislation et rxc3xa9glementationsxe2x80x94Produits cosmxc3xa9tiques et produits d""hygixc3xa8ne corporellexe2x80x941997xe2x80x94page 229).
Finally, the water present in these cosmetic and dermo-cosmetic bases constitutes a very favourable environment for radical reactions and, in particular, oxidation reactions. Thus, the incorporation of molecules very sensitive to oxidative stress, such as vitamin C, certain fatty acids or certain polyunsaturated molecules, is very difficult due to low stability in time.
In the case of bases associating an aqueous phase and a lipidic phase, the essential presence of amphipilic molecules with surface-active powers allows stable dispersion and the blending of the two phases. These molecules may be rather aggressive for the skin (Les molecules de la beautxc3xa9. De l""hygixc3xa8ne et de la protection pages 33-38, les tensioactifs, Pierre le Perchec, Ed. Nathan). We are therefore seeking to obtain anhydrous bases in which the active principles can be incorporated at temperatures less than 80xc2x0 C. and whose texture is sufficiently creamy to allow satisfactory spreading with the fingers.
Anhydrous formulations destined for cosmetic and dermo-pharmaceutical applications have been known for a long time. The oldest are constituted from animal, vegetable, or mineral oils, or a blend of these. These oily forms are no longer used due to their greasy texture. Makeup bases in greasy phase are used above all because they adhere to the epidermis, provide protection and form a water-impermeable film. Anhydrous makeup products often come in a solid compact form, for example, lipstick, or in cream form (Peau-Soins cosmxc3xa9tioques et Prxc3xa9parations dermo-pharmaceutiquesxe2x80x94R. Raoult, Ed. Porphyre page 106).
These bases, whilst interesting, are, nonetheless, very greasy and thick. On the one hand, their stability implies the presence of waxes whose melting temperatures, generally higher than 80xc2x0 C., prevent the use of thermo-sensitive active principles.
An improvement is proposed in the French patent FR 2 734714, with the use in the base of an association of, in particular, organo-modified clay, polymer particles, pyrogenic silica dispersed in a greasy phase without the use of wax. This base, in anhydrous gel form, though still greasy is sufficiently creamy to be spread with the fingers. It can incorporate thermo-sensitive molecules. However, the clays used are modified by greasy quaternary ammonium salts that can be irritating for the skin. Finally, the greasy phase comprises oils of an amphiphilic nature, giving a shiny aspect to the skin.
After different studies, we have noted that the inconveniences of such previously used bases, that is to say:
the drying effect, the presence of surface-active agents and antioxidants, the impossibility to use molecules sensitive to the oxidative stress in the case of aqueous bases,
the greasy effect, the impossibility of using thermo-sensitive molecules, the shiny aspect and the difficulty of spreading for the anhydrous bases,
could be solved by the use of pearls with a strictly anhydrous base and completely lacking surface active-agents, thus defining the invention.
Surprisingly, we have discovered that the association of oils, mineral waxes, animal or vegetable waxes, talc and silica, give a solid base, stable in time, able to contain active principles, offering a non-greasy, melting, film-forming, easy-to-spread texture without residues nor a shiny aspect.
However this base is not creamy enough to be easily taken with the fingers from ajar or tube container.
We have discovered that the particles prepared from this base, used alone, adhere sufficiently to the epidermis to be able to be taken easily with the finger and to be applied whilst retaining the properties of the base. We will give the name pearls to the particles resulting from the shaping of these bases. The base appears to be in a dispersed state, allowing topical cosmetic and dermo-pharmaceutical applications.
One of the aspects of this invention is that the base comprises at least one hydrophobic wax, a non amphiphilic oil and talc. The final melting temperature must be between 15xc2x0 C. and 70xc2x0 C., and preferably between 20xc2x0 C. and 45xc2x0 C.
The invention bases are mainly constituted from wax or blends of mineral waxes, non-greasy, non-amphiphilic mineral oils, talc and silica. They can contain, in addition, oily, paste or solid additives and liposoluble or hydrosoluble active ingredients.
The base generally contains from 0.1% to 40% of paraffin wax or a blend of paraffin wax and waxes chosen preferably from among:
Carnauba wax
Candelilla wax
Alfa wax
ozocerite
vegetable waxes such as olive wax, rice wax, hydrogenised jojoba wax or absolute flower waxes.
bees"" waxes or modified bees"" waxes.
It is possible to use other wax matrices, but the blend obtained must be characterised by a melting point of less than 90xc2x0 C., by the absence of surface-active substances and a hydrophobic behaviour, and be non-wettable by water.
In the wax blend, paraffin wax represents, preferably, at least 40% of weight in relation to the total weight of the wax blend.
In addition to the waxes indicated above, the invention base generally contains a mineral oil or a blend of mineral oils, preferably a paraffin oil or a blend of paraffin oils and mineral oils, or synthetic oils chosen among:
hydrophobic silicone oils with viscosity of between 5 and 9000 centistokes and more particularly:
cyclomethicones
lipophilic organofluorinated oils
perhydrosqualen
In the oil blend, paraffin oil represents, preferably, between 4% and 90% in weight in relation to the blend. Other oily compounds, such as oleic alcohol, lanolin, sunflower oil, palm tree oil, can be used, but the oily blend obtained must be characterised by hydrophobic behaviour, must not be miscible in water and must have a melting point of between 0xc2x0 C. and 45xc2x0 C.
In addition to the oils and waxes indicated above, the invention base contains talc, sometimes with the addition of silica. We choose preferably a talc with a fine granule size, less than 5 micrometers and a pyrogenic silica that has not undergone any chemical derivation. The base generally contains 0.5% to 10% in weight of talc and silica particles. Moreover, it is possible to adjust the consistency by adding to the base, clays or their oily dispersions, phenylated silicon gums, starches and fatty structuring substances.
A particularly preferred form of the invented base also contains a polyoxyethylene glycol. Its addition allows the soft and melting qualities of the bases to be improved.
The invention bases are obtained by mixing at moderate heat. More precisely, these bases are obtained by a procedure characterised by mixing the wax and oil, at wax melting temperature, until obtaining a blend with a melting temperature less than the wax melting temperature. The initial relationship between the wax and the oil can be modulated so that the melting temperature of the final blend is less than the temperature at which the most heat-sensitive substance to be incorporated deteriorates. In practice, the percentage in weight of hydrophobic wax in the blend is between 1 and 40%. The final blend must be solid at room temperature and preferably have a melting temperature of 30xc2x0 C. The blend is then cooled by adapted agitation, at a temperature 2xc2x0 C. or 3xc2x0 C. higher than its melting point, to allow the inclusion of the cosmetic or pharmaceutical active ingredients, silicon oils, polyoxyethylene glycols, talc and silica.
The compounds obtained are then shaped to give spherical hydrophobic particles called pearls.
One aspect of this invention is, therefore that it makes it possible to obtain an anhydrous base without emulsifying or surface-active agents. This invention also concerns a procedure for the preparation of pearls from bases as defined above.
The main techniques for preparing spheroid particles are mainly mechanical. For example, hot granulation around a solid core in a turbine or by a bed of fluidised air or in a planetary mixer, by passage through a channel, by moulding, by casting, or injection, hot or under pressure, in the moulds. These techniques have all the inconvenience of requiring important equipment and not allowing easy modulation of the size of the particles. The procedures are long and the base is subject to thermal, mechanical and oxidative stress, in the case of hot shaping under pressure.
The solvent evaporation technique, described in the patent FR 0 505 648, is based on the dispersion of an organic phase containing the constitutive products of the particles and the dispersing agents, in an aqueous non-miscible phase under agitation. This phase is followed by the elimination, without agitation, of the organic solvent under reduced pressure allowing the formation of particles. This method does not make it possible to obtain large-sized particles. Moreover, the complete elimination of the solvent is very difficult and finally, the particles obtained contain all or part of the dispersing agent.
The techniques based on extrusion and/or crushing imply the formation of particles with irregular surfaces and subject the base to thermal, mechanical and oxidative stresses.
Less aggressive techniques, or those that require little heating, exist for the treatment of substances sensitive to heat, oxidation, and mechanical stress. These are so-called cold solidification methods.
In the description of patent EP 0 438359, the cold blend containing the active ingredients and excipients, is dispersed in the form of droplets, by its passage through a nozzle, in a tower where they are cooled by a cold gas in counter flow, then recovered. Whilst this technique presents a clear improvement over the preceding techniques, it remains complex to implement, the dispersion stage is difficult to control and the size of the particles is not easily varied. We cannot obtain, or only with difficulty, particles whose size is greater than one millimetre. Finally, this technique does not allow large yields.
The techniques based on the cooling of the melted base, maintained in movement in a non-solvent liquid such as water, described in patents FR 2 705232, FR 2 660 534, EP 0 0435 008, are referred to as hot-melting techniques.
These techniques described by Langer (E. Mathiowcz and R. Langer, Journal of Controlled Release, 5. 1987, pages 13-22), consist in dispersing the compound or blend, previously melted, in a liquid heated to the same temperature, in which it is not miscible. The droplets obtained are solidified by cooling of the dispersion environment during agitation. As any professional will know, one of the great difficulties in these procedures is to obtain a good stabilisation of the emulsion produced. In fact, if the agitation is insufficient, the droplets tend to merge to reconstitute the two phases. The emulsion must be stabilised without variations in the size of the droplets until cooling at the solidification temperature. The size of the droplets depends on the speed of agitation, the viscosity of the dispersion environment and, of course, the presence of surface-active or emulsifying agents. In patents FR 2 658829 and EP 0 045008, the dispersion is stabilised by the presence of amphiphilic compounds allowing the stabilisation of the dispersion in aqueous phase and by maintaining important agitation. Whilst these procedures are interesting, they do not make it possible to obtain particles of sizes exceeding 1,000 micrometers and the elimination of surface-active agents remains very difficult.
In the description of patent FR 2 660554, the procedure does not call on the use of any emulsifying agent, since the lipidic base contains amphiphilic compounds allowing the dispersion to be stabilised in aqueous phase. It is not possible with such bases to prepare particles larger than 1000 micrometers by this procedure, and the particles thus prepared can hydrate and absorb up to eight times their weight in water. This procedure is therefore not compatible with strictly hydrophobic bases.
In the description of the patent FR 2 705232, the active ingredient is added to the hydrophobic polymer-based base at the time of the dispersion in an aqueous phase at a temperature exceeding the vitreous transition temperature. This procedure has the inconvenience of being long, since hot dispersion must be maintained until the end of the encapsulation of the active ingredients by the droplets.
This invention proposes a new procedure for the preparation of particles from bases part of the invention, in which the inconveniences of the techniques described in the preceding procedures can be avoided.
In comparison with the procedures of the aforementioned patents, the procedure of the present invention is carried out without using any emulsifying agent, solvent or pre-shaped particles and it is much more rapid. The particles obtained in this invention are spherical and between 1 and 10,000 micrometers in size. According to the invention""s preferred method of realisation the pearls are between 1,000 and 10,000 micrometers in size and contain, dissolved or dispersed in their matrix, cosmetic, pharmaceutical or biotechnological ingredients. According to this method, the lipidic base of the pearls serves as a support and vehicle for ingredients such as perfumes, essential oils, aromas, pigments, loads, colorants, enzymes and coenzymes and other active cosmetic substances. The loading capacity of the pearls can vary from 0.02% to 75% in relation to the pearl weight.
As professionals will know, when we incorporate these constituents into the pearls, we must choose an appropriate lipidic base so that the procedure can be implemented, and that the pearls are solid at room temperature and preferably between 1,000 and 10,000 micrometers in size.
Among the cosmetic constituents that can be incorporated into the pearls we can cite vitamins or provitamins A, B, C, D, E, PP and their esters, hydrants, melanin, carotenoids, anti-radical substances, U.V. filters, keratolytic substances such as salicylic acid and its salts, molecules acting on pigmentation, inflammation, softening agents such as fatty esters, sebum substitutes or constituents, biological extracts, hair dyes, antiperspirants, molecules capable of trapping odours and aromas, so-called slimming molecules. Other constituents can be incorporated into the pearls, such as softening or lubricating substances for the skin, for example talc, kaolin, mica, nano-titanium, polyamide polymer micro-balls, or, to the contrary, powders destined for abrasion using particles of silica, or of polymer or plant origin.
The pearls can also contain preservatives, antioxidants, colorants and pigments along with dulling agents such as, for example, magnesium carbonate, stock, zinc power, zinc oxide, Toshiba Tospearl silicon blend micro-balls. We can incorporate into these pearls other microparticles or microcapsules, vector or matrix systems or substances containing active principles, cells and cell organites. Finally these pearls can contain pharmaceutical constituents destined to treat skin or mucous pathologies or capable of trans-dermal diffusion.
In this description, the term active constituent is used to refer to any active therapeutic substance or blend that can be beneficially administered to man or other mammal to diagnose, tend, reduce, treat or prevent disease. We can give the examples of antibiotics, hormones and derivatives, nicotine, anti-histamine agents, steroid and non-steroid anti-inflammatory agents, anti-allergic agents, local anaesthetics, vasodilators, anti-viral agents, antibodies and molecules acting on the immune system. This long list is not exhaustive.
In the case of oral administration of this type of particle, an appropriate base must be chosen, compatible in terms of toxicity, biocompatibility and bio-degradability with oral absorption. In this case the ingredients will be chosen from among the ingredients already used for oral administration and in such a way that the pearls formed retain their properties of incorporation and stabilisation of active constituents.
The pearls in this invention can contain mineral or organic pigments, including pearly pigments. Among the organic pigments, we can cite carbon black, varous D and C Red organic pigments, orange or yellow coded in the colour index. Among the mineral colorants, we can cite titanium dioxides, iron oxides, brown, black or yellow, chromium oxides, silicate of aluminium polysulphide ultramarine, manganese pyrophosphate, Prussian blue or ferric ferrocyanide. The pigments are present in the pearls in concentrations of between 0 and 30% and preferably between 1 to 20% in weight in relation to the pearl weight.
The pearls in this invention, loaded with pigments, can be used as base for makeup products such as foundations, complexion refiners, makeup correctors, tinted creams, eye shadow, blushers and lipsticks.
One aspects of the present invention is that it offers a procedure for preparing pearls containing pigments. The pearls containing pigments, prepared according to the invention procedure, can be used as a base for cosmetic products. In this preparation method, pearls can be less than 10 micrometers in size. In these conditions, the pearls can be used for the incorporation of pigments destined for the preparation of paints, inks and dyes.
This invention provides a new procedure for the preparation of particles called pearls in which the inconveniences of the techniques described in the procedures recalled above can be avoided.
In comparison with the hot melting techniques, the procedure of the present invention does not call on the intervention of emulsifying agents or amphiphilic products in the base to allow a stable dispersion at the time of the solidification by cooling phase. The blend of the different constituents and active principles forming the compound from which the pearls will be obtained is carried out in the first stage of the procedure. The mixing is carried out under heat, 2xc2x0 C. or 3xc2x0 C. above the melting points of the constituent presenting the highest melting point. As any professional will know, it is necessary to apply the agitation that is appropriate to the dispersion of all the constituents. Then the compound droplets are formed by dispersing the compound in a gel, previously heated to the same temperature, with which it is not miscible, with a sufficiently high viscosity to fix the dispersion. It is preferably to inject the compound within the gel, for example by a hole located at the base of the reactor. Agitation must be maintained throughout the injection and is carried out in an agitator system with blade equipped with an anchor, destined to disperse the compound, and a second axial blade equipped with a 3-blade propeller destined to form dispersion droplets of the desired size. This last stage is extremely rapid since the droplets are obtained gradually as the compound is injected. Agitation does not have to be maintained, since the droplets are fixed in the gel. At the end of injection, they are immediately cooled to the solidifying temperature then washed and recovered dry.
This procedure is therefore rapid and does not require any long and delicate agitation stage. It makes it possible to incorporate the active principles in the base right at the first phase of the mixing of the different compound products.
Among the gelling agents suitable for forming the gels used for the dispersion environment in the invention, we can cite carboxyvinyl polymers such as carbopols neutralised by soda, carrageen, polysaccharide thickeners and gelling agents such as xanthenes, guar and carob gums, alginates, cellulose, pectin and agar derivatives.
The gels used in the pearl preparation procedure have a concentration generally of between 0.1% and 50% in weight in relation to the total weight of the gel.
The pearls obtained are extremely homogeneous in size and can be manipulated industrially without special precautions. The following examples are not exhaustivexe2x80x94they are just to illustrate the invention

Cosmetic or dermopharmaceutical composition in the form of beads and methods for preparing same